Post-translational modifications are essential regulators of protein function in cells. In many cases, the mechanisms and substrate specificities of enzymes that catalyze these modifications are not well understood, despite their involvement in a multitude of diseases, including cancer, diabetes, cardiovascular disease and neurological disorders. Our studies will focus on two classes of post-translational modifications, lipidation (including prenylation and palmitoylation) and acetylation, and the enzymes that regulate these modifications. The majority of protein prenylation, whereby an isoprenoid group is appended near the C-terminus of a target protein, is catalyzed by protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I). Palmitoylation is catalyzed by protein palmitoyl transferases (PATs), such as Akr1p, and is a thioester exchange reaction between palmitoyl-CoA and a cysteine on a substrate protein to form a palmitoyl thioester. Both prenylation and palmitoylation target proteins to membranes and are essential for protein trafficking. Protein acetylation is regulated by competition between histone acetyltransferases, that catalyze protein N-acetylation, and histone deacetylases (HDACs), that catalyze hydrolysis of 5-N-acetyl lysine. Acetylation affects multiple cellular processes, including gene expression and cell differentiation. All of these enzymes are current targets for drug development for a variety of diseases; HDAC inhibitors are approved for clinical use and FTase and GGTase inhibitors are currently in clinical trials. However, the full complement of in vivo substrates for these enzymes is unknown, and the functional basis for substrate selectivity by each enzyme is undefined. Moreover, the effects of these modifications on protein function and cellular pathways are frequently unclear. Therefore, significant questions remain for each enzyme, including their in vivo specificity and regulation. Here, we propose to: (1) investigate determinants of prenyltransferase substrate specificity for mammalian and Candida albicans enzymes and analyze the effects of individual prenylation pathway modifications on protein localization and function; (2) investigate the catalytic mechanism of the protein palmitoyltransferase Akr1p; (3) investigate the metal selectivity of deacetylases HDAC8 and LpxC in vivo and in vitro and the potential for regulation by metal switching in response to metal availability and redox stress; and (4) identify substrates and evaluate determinants of HDAC8 and HDAC11 substrate specificity, both in vitro and in vivo, by chemical crosslinking and activity-based probes. Our long-term goals are: (1) to determine the catalytic mechanism and molecular basis of specificity for each of these post-translational modifying enzymes; and (2) to examine the extent, biological function and regulation of prenylation, palmitoylation, and acetylation in the cell. Overall, our studies will assist in targeted drug design by describing the specific interactions that dictate substrate specificity and by identifying novel regulatory pathways as targets for disease therapies.